1. Field of the Invention
This invention relates generally to methods of forming nanoscale structures and the nanoscale structures formed thereby. In particular, this invention relates to methods using multi-photon excitation for the fabrication of structures with nanometer-level precision.
2. Description of the Related Art
Three-dimensional objects having fine-scale microstructures possess unique and technologically attractive properties. There has been particular interest recently in the fabrication of structures with nanometer-level precision, that is, objects with structures or structural features measurable in the nanometer range. Such nanoscale structures have dimensions or features in the range of about 2 to about 100 nm (nanometer, wherein 1 nm=10 angstroms), which is on the order of the size of macromolecules such as proteins and protein complexes.
Photolithography, including methods using X-ray and deep UV, is well-known for producing two-dimensional structures with small-scale features. However, this technique does not allow the production of complex, curved three-dimensional surfaces, as it is very limited in the complexity achievable in the z-direction. Three-dimensional objects produced by photolithographic methods have therefore been essentially limited to columnar structures. Objects with features smaller than 150 nm are not readily producible or routinely available. George M. Whitesides has also described several methods for micro-scale fabrication based on microcontact printing and modification of surface chemistry with self-assembled monolayers. These methods, however, are also very limited in the ability to build in the third dimension, as well as in their chemistry. A method for manufacturing three-dimensional optical data storage and retrieval structures by reaction of polyesters using two-photon excitation is disclosed in U.S. Pat. No. 5,289,407 to Strickler, et al., which is incorporated herein by reference in its entirety.
A number of other, different approaches have been described for the synthesis of three-dimensional objects with small-scale features, for example biomimetic matrix topographies such as basement membrane textures. As described in U.S. Pat. No. 5,700,241, such structures are produced by removal of epithelial or endothelial cell layers to expose the supporting basement membrane or matrix. The exposed topography is then used as a mold for polymer casting. The surface of the resultant molded negative replica of the matrix topography is then itself cast with the final (bio)material of choice. With this methodology, three-dimensional biomimetic matrices can be prepared for both experimental investigations into cellular biology, and to potentially improve the cell and tissue response to implanted biomaterials. Although this method can produce very complex three-dimensional topography, it does not provide for topographic design flexibility, since all constructs must begin with a biological surface. In addition, while many materials may be used for fabrication, the procedure does not provide for spatial control of chemistry.
Scanning tunneling microscopy has also been used to move atoms on surfaces. However, this technique is extremely limited in the sizes and chemistry of the fabricated region. Another technique which has been described for the solid, free-form fabrication of microscale structures includes forming successive, adjacent, cross-sectional laminae of the object at the surface of a fluid medium or other bed, the successive laminae being automatically integrated as they are formed to define the desired three-dimensional object, as disclosed in U.S. Pat. No. 4,575,330 to Hull. U.S. Pat. No. 5,518,680 to Cima et al. similarly discloses successive printing of layers of powder in a solvent which causes binding of the successive layers, thereby allowing the formation of drug delivery devices having thicknesses on the order of about 100 microns.
Three-dimensional objects have also been generated by selective curing of a reactive fluid medium by a beam or beams of ultraviolet (UV) radiation brought to selective focus at prescribed intersection points within the three-dimensional volume of the fluid medium. Disadvantages of such systems include the use of UV radiation, which requires expensive and cumbersome optics and lens, as well as the associated poor focusing qualities of excimer and other UV laser sources.
An additional technique for generating three-dimensional microscale objects is described by S. Maruo, O. Nakamura, and S. Kawata et al. in xe2x80x9cThree Dimensional Microfabrication With Two-Photon-Absorbed Photopolymerizationxe2x80x9d, Optics Letters, Vol. 22, No. 2, pp. 132-134 (1997), which is incorporated herein by reference in its entirety. Maruo et al. discloses that microscale structures are formed by subjecting urethane acrylate monomers and oligomers to near-infrared laser light in a non-solvent system. Use of two-photon absorption for initiation of the reaction leads to a spiral wire having a diameter of 6 microns, an axial pitch of 10.3 micron, and a width of nearly 1.3 microns. While small, such structures are not in the nanoscale region. Maruo et al. furthermore only describe synthesis in a non-solvent system, which is incompatible with most biomolecules.
Accordingly, there still remains a need for methods of free-form fabrication of two- and three-dimensional structures having dimensions or features in the micron and nanometer range, especially techniques suitable for synthesis using biomolecular subunits such as proteins, peptides, oligonucleotides, as well as bio-active small molecules such as hormones, cytokines and drugs.
The above-discussed and other drawbacks and deficiencies of the prior art are overcome or alleviated by the method and apparatus of the present invention, wherein small, two- or three-dimensional structures are formed by multiple-photon-absorbed photopolymerization and/or cross-linking of a precursor composition, that is, photopolymerization using multi-photon excitation. Use of multi-photon excitation allows fabrication of structures and structural features having at least one dimension of less than about one micron, preferably less than about 500 nm, more preferably less than about 250 nm, and most preferably of less than about 100 nm, in bulk phase as well as in solution, and from a wide variety of organic and inorganic precursor subunits, including synthetic polymers and biological polymers such as proteins, lipids, oligonucleotides, and the like.
In one embodiment, use of two-photon far field optics allows the formation of structures having X-Y dimensions of less than about 300 nm and a Z dimension of less than about 500 nm, while use of three-photon far field optics allows the formation of structures having X-Y dimensions of less than about 250 nm and a Z dimension of less than about 300 nm. In a particularly preferred embodiment, use of a 4 pi optical configuration in combination with two-photon far field excitation allows the formation of structures having X-Y dimensions of less than about 150 nm and a Z dimension of less than about 100 nm. In another embodiment, use of multi-photon near field optics results in the formation of structures having X, Y, and Z dimensions of less than about 50 nm. In this embodiment, near field fabrication is achieved by two-photon excitation through fiber probes. In a related embodiment, the optical element of the near field embodiment is coupled with a multiple barreled pipette for precise delivery of components into multiple areas simultaneously or sequentially.
The method described herein is useful for the formation of a variety of small-scale structures. In one embodiment, noncross-linked agents are entrapped (permanently or temporarily) in a gel or matrix formed by multi-photon excitation. Such gels or matrices may have controlled release, degradation, and/or diffusivity properties. Agents include proteins, peptides, carbohydrates, drugs, enzymes, liposomes, nucleotides, and cells. In a related embodiment, multi-photon excitation is used to fabricate devices having varying cross-link densities and/or chemistries to produce materials having variable degradation properties for use as controlled release devices in drug delivery, biomaterials, tissue engineering, and environmental applications.
In another embodiment, multi-photon excitation is used to modify the surface of biological or conventionally fabricated materials. The materials may have complex surface features, or the present method may be used to provide complex features to the materials. Exemplary applications include adding one or more bioactive functions to an integrated circuit (IC) chip, manufacturing biomimetic surfaces for use with tissue cell culture, and modifying explanted tissue for re-implantation or other uses. The combination of microscopy and multi-photon excitation allows the micro-positioning of one or more features on a surface. In a related embodiment, multi-photon excitation is used to manufacture ciliated surfaces or other micro-sized transport devices using motile proteins.
In another embodiment, multi-photon excitation is used to create structures which, in conjunction with shrinkage or expansion effects, dynamic shape change effects (i.e., Poisson ratio effects), and/or groups active under certain chemical conditions, will result in more complex structures. Such structures may be used as a variable filter or as a small-scale actuator to exert physical force, alter fluid flow, and the like. In a related embodiment, optical devices are manufactured in layers and/or in other two- and three-dimensional configurations by configuring optically active and chiral compounds.
In another embodiment, multi-photon excitation is used to provide spatial orientation of enzymes on or within substrates or manufactured constructs. Organization is provided by application of electrostatic fields, selective adsorption, shear forces and by optical and magnetic traps.
In another embodiment, proteins are cross-linked directly, without use of photosensitizers or chemical crosslinking agents.
In another embodiment, multi-photon excitation is used to effect nanofabrication at remote sites via optical fibers. Such optical fibers may be placed, for example inside a catheter. Nanofabrication in this embodiment includes delivery of drugs or other biologically active agents, controlled delivery of tissue engineering scaffolding agents, growth factors, and the like; and minimally invasive assembly of structural elements or devices such as stents.
The method and apparatus of the present invention allows the formation of structures having smaller dimensions than before possible. The above-discussed and other features and advantages of the present invention will be appreciated and understood by those skilled in the art from the following detailed description and drawings.